DETECTION OF ENERGY-TRANSFER BETWEEN TRYPTOPHAN RESIDUES IN THE TUBULIN MOLECULE AND BOUND BIS(8-ANILINONAPHTHALENE-1-SULFONATE), AN INHIBITOR OF MICROTUBULE ASSEMBLY, THAT BINDS TO A FLEXIBLE REGION ON TUBULIN
DETECTION OF ENERGY-TRANSFER BETWEEN TRYPTOPHAN RESIDUES IN THE TUBULIN MOLECULE AND BOUND BIS(8-ANILINONAPHTHALENE-1-SULFONATE), AN INHIBITOR OF MICROTUBULE ASSEMBLY, THAT BINDS TO A FLEXIBLE REGION ON TUBULIN
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DOI：
10.1021/bi00360a010
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发表时间：
1986-06-17
期刊：
影响因子：
2.9
通讯作者：
HOROWITZ, PM
中科院分区：
文献类型：
--
作者：
PRASAD, ARS;LUDUENA, RF;HOROWITZ, PM
The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 .mu.M, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37.degree. C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin-Weber plots were obtained from bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated form Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.
